Recently, we have made the novel and serendipitous observation that nicotine is an extraordinarily potent agent of angiogenesis [1]. Our studies indicate that endothelial nicotinic acetylcholine receptors (nAChR) mediates this effect of nicotine. Whereas there is a substantial body of literature regarding the receptor composition, distribution and signaling of neuronal nAChR, there is little known about signaling in the recently discovered endothelial nicotinic cholinergic pathway. Furthermore, there is no information about how this pathway is recruited by angiogenic stimuli (e.g. hypoxia). Accordingly, our specific aims are to: 1) Characterize the effect of hypoxia on the expression of nAChR and the intracellular machinery for synthesizing and releasing ACh from endothelial cells, including expression and activities of the choline transporter, choline acetyltransferase, and acetylcholinesterase. We will identify the nAChR subunits that are expressed on endothelial cells and their modulation by hypoxia or VEGF. We will use a variety of genetic or pharmacological tools to knock down the expression or activities of putative elements in the nAChR pathway, and observe the functional effect on calcium flux, NO elaboration and EC migration. 2) Characterize the signaling pathways activated by stimulation of endothelial nAChR(s), focusing on those that are also known to be involved in NO synthase activation (as NO appears to be a critical mediator of nAChR-induced angiogenesis). To dissect out the role of various signaling proteins, we will use Western analysis, kinase activity assays and observe the effect of specific pharmacological antagonists, dominant negative mutants or RNAi on calcium flux, NO elaboration and EC migration. We will identify genes uniquely regulated by nAChR(s) using subtraction suppression hybridization and DNA microarray, and further characterize the time course, dose response, and endothelial selectivity of expression, with the assistance of analytical tools including SAM, GeneMAPP, hierarchical clustering to transform the raw microarray data into useful information. Of the regulated, endothelial-selective nicotine-specific genes that are identified, initially one of the most promising candidates will become the target of in vitro mechanistic studies designed to elucidate the role of the gene in the angiogenic effects of the nAChR(s).